Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle

Background Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne’s disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne’s disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. Results Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. Conclusions This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne’s disease progression by warranting further research on the presence of MAP in blood

Salmonella Strains Isolated from Galápagos Iguanas Show Spatial Structuring of Serovar and Genomic Diversity

It is thought that dispersal limitation primarily structures host-associated bacterial populations because host distributions inherently limit transmission opportunities. However, enteric bacteria may disperse great distances during food-borne outbreaks. It is unclear if such rapid long-distance dispersal events happen regularly in natural systems or if these events represent an anthropogenic exception. We characterized Salmonella enterica isolates from the feces of free-living Galápagos land and marine iguanas from five sites on four islands using serotyping and genomic fingerprinting. Each site hosted unique and nearly exclusive serovar assemblages. Genomic fingerprint analysis offered a more complex model of S. enterica biogeography, with evidence of both unique strain pools and of spatial population structuring along a geographic gradient. These findings suggest that even relatively generalist enteric bacteria may be strongly dispersal limited in a natural system with strong barriers, such as oceanic divides. Yet, these differing results seen on two typing methods also suggests that genomic variation is less dispersal limited, allowing for different ecological processes to shape biogeographical patterns of the core and flexible portions of this bacterial species' genome

STABILIZATION OF COBALT CAGE CONFORMERS IN THE SOLID-STATE AND SOLUTION

The cage complexes [Co{(NMe(2))(2)sar}](3+) and [Co{(NMe(2),Me)sar}](3+) (sar = sarcophagine = 3,6,10,13,16,19 hexaazabicyclo[6.6.6]icosane; (NMe(2))(2)sar 1,8-bis(dimethylamino)sarcophagine; (NMe(2),Me)sar 1-(dimethylamino)-8-methylsarcophagine) are obtained by methylation of amino substituents on the parent cage complexes using formaldehyde and formic acid. Further methylation with methyl iodide in dimethyl sulfoxide, or with dimethyl sulfate in N,N-dimethylformamide, converts the dimethylamino substituents to trimethylammonium substituents and essentially inverts the circular dichroism (CD) spectrum in comparison to that of the protonated parent cage complex, [Co{(NH3)(2)sar}](5+). A combination of NMR and electronic spectroscopic measurements indicates that the inversion of the CD spectra is due to conformational inversions in the 1,2-ethanediamine rings from mainly a lel conformation in [Co{(NH3)(3)sar}](5+) to an obs conformation in [Co{(NMe(3))(2)sar}](5+), (without inversion at any of the seven stereogenic centers; Co plus six coordinated amines). This ob(3) conformation was identified in the solid state by an X-ray crystallographic analysis of [Co{(NMe(3))(2)sar}](NO3)(5).3H(2)O: monoclinic, P2(1)/n, a 14.382(4) Angstrom, b = 14.604(3) Angstrom, c 16.998(6) Angstrom, beta = 100.28(3)degrees, Z= 4. The electron self-exchange rate constant of the [Co((NMe(3))2sar)](5+/4+) redox couple (+0.05 V vs NHE, 0.011(1) M(-1) s(-1) at 25 degrees C, I = 0.2 M (NaCl)) is a factor of 2 smaller than that of the [Co((NH3)2sar)](5+/4+) couple under the same conditions and is the slowest electron-transfer rate so far measured for a hexaamine cage complex of this type. However, it is still much faster than those of the parent [Co(en)(3)](3+/2+) and [Co(NH3)(6)](3+/2+) couples. This paper also provides hard evidence for the conformations of other Co(III) cage complexes in solution as well as the solid state